Different culture conditions used for arresting the G0/G1 phase of the cell cycle in goldfish (Carassius auratus) caudal fin-derived fibroblasts

One of the most important factors determining the success of the development of cloned embryos is the cell cycle stage of the donor cells. We investigated the effects of serum starvation, culturing to confluence and roscovitine treatment on the cell cycle synchronization of goldfish caudal fin-derived fibroblasts by flow cytometric analysis. The results show that culturing the cells to confluence (85.5%) and roscovitine treatment (82.71%) yield a significantly higher percentage of cells arrested in the G0/G1 (P < 0.05) phase than serum starvation (62.85%). Different concentrations of roscovitine (5, 10, or 15 μM) induce cell cycle arrest at the G0/G1 phase.     

Differential protection against oxidative stress and nitric oxide overproduction in cardiovascular and pulmonary systems by propofol during endotoxemia

Background  

Both overproduction of nitric oxide (NO) and oxidative injury of cardiovascular and pulmonary systems contribute to fatal cardiovascular depression during endotoxemia. We investigated in the present study the relative contribution of oxidative stress and NO to cardiovascular depression during different stages of endotoxemia, and delineated their roles in cardiovascular protective effects of a commonly used anesthetic propofol during endotoxemia.     

Properties of biodegradable citric acid-modified granular starch/thermoplastic pea starch composites

Pea starch-based composites reinforced with citric acid-modified pea starch (CAPS) and citric acid-modified rice starch (CARS), respectively, were prepared by screw extrusion. The effects of granular CAPS and CARS on the morphology, thermal stability, dynamic mechanical thermal analysis, the relationship between the mechanical properties and water content, as well as the water vapor permeability of the composite films were investigated. Scanning electron microscope and X-ray diffraction reveal that the reinforcing agents, the granules of CAPS and CARS, are not disrupted in the thermoplastic process, while the pea starch in the matrix is turned into a continuous TPS phase. Granular CAPS and CARS can improve the storage modulus, the glass transition temperature, the tensile strength and the water vapor barrier, but decrease thermal stability. CARS/TPS composites exhibit a better storage modulus, tensile strength, elongation at break and water vapor barrier than CAPS/TPS composites because of the smaller size of the CARS granules.     

46,XY女性性反转患者SRY基因的两个新突变类型

Y染色体上的性别决定区域——SRY基因作为睾丸决定因子,可以调控男性性别发育过程。SRY基因是一种转录因子,属于带有高迁移率族蛋白家族,该家族成员包含能与DNA结合的HMG盒基序。已知SRY基因的缺失和点突变是造成XY女性性反转的病因之一。通过筛查10位中国46,XY女性性反转病人SRY基因的开放阅读框区域,探寻新的突变类型。用标准方法从外周血中抽提gDNA,通过聚合酶链式反应扩增SRY基因中部的609bp的DNA片段。扩增后的PCR片段被克隆到pUCm-T载体中,在ABI377-3自动测序仪上完成测序。运用限制性内切酶酶切分析的方法验证DNA测序的结果。结果表明,在两个患者的SRY基因中分别发现了新的核苷酸点突变,并都导致氨基酸替代。一个突变发生在SRY基因的5’端HMG盒外的核苷酸第113位腺嘌呤(A)被鸟嘌呤(G)取代,并导致谷氨酸被甘氨酸替换;另一个突变是第387位核苷酸发生T被A替换,该突变引起第129位的酪氨酸变成终止密码,她父亲的SRY序列被证明是正常的野生型。通过查询文献和人类基因突变数据库(HGMD),这两个突变都是以前未见报道过的新型SRY基因突变,并使因核苷酸替换引起SRY基因突变总数增加到45。     

系统发生分析发现牛病毒性腹泻病病毒新基因亚型

本研究对我国首次分离获得的牛源牛病毒性腹泻病毒(BVDV)毒株Changchun 184(CC-184)和猪源牛病毒性腹泻病毒ZM-95进行了遗传衍化关系研究.选择主要抗原E2基因为研究对象,首先应用RT-PCR及套式PCR克隆得到CC-184和ZM-95的E2片段,通过序列测定发现CC-184和ZM-95 E2基因长度分别为1,122bp和1,125bp,各自编码374和375个氨基酸残基.核酸序列同源性比较和系统发生分析表明2株病毒均属于BVDV-1,CC-184与Osloss亲缘关系最近,都属于已有的b基因亚型,其E2基因同源性达91.8%.而ZM-95的E2基因有一个特征性的变异区,包含一个密码子序列插入,这一变异区编码了一段有别于其他瘟病毒的五肽氨基酸序列HYKKK.结果还表明ZM-95与BVDV-1现有的5个基因亚型的亲缘关系均较远,E2基因同源性最高(与Oregonc24v)只有72.4%.而BVDV 1亚型内毒株间的同源性大于85%,亚型间的同源性在69%～75%之间,充分说明ZM-95是BVDV-1中一个新发现的基因亚型.通常认为猪源BVDV来源于牛,应该与牛源BVDV有十分近的遗传关系,但是本研究发现ZM-95与其他已知牛源BVDV较低的基因同源性说明猪源BVDV还具有独立的遗传衍化与传播来源的可能性.     

巴斯德毕赤酵母表达的突变型人白细胞介素-2的发酵条件与纯化研究

在以前的研究中,通过蛋白质工程技术获得了三突变体白细胞介素_2基因(编码125位半胱氨酸→丙氨酸;18位亮氨酸→蛋氨酸;19位亮氨酸→丝氨酸) ,并在毕赤酵母中加以表达。进一步优化表达条件,其最适诱导条件:80 %以上的通气,诱导2d ,初始pH60 ,甲醇终浓度为10%。在上述条件下表达量占菌体总蛋白的30%以上,大约200mg L。建立了一套从毕赤酵母表达上清中分离纯化分泌型表达蛋白IL_2的方法,经离心,超滤浓缩,强阳离子交换S柱和分子筛层析得到纯化的突变型和野生型IL-2 ;其得率为27% ,纯度达电泳纯并且HPLC检测只有一个峰。纯化的突变蛋白对CTLL-2细胞具有刺激性;与野生型IL-2相比,在各种温度条件下储存的突变蛋白保留有更高的活性;突变型IL-2的活力是野生型的4～5倍,具有更高的利用价值。     

Synthesis and antiproliferative evaluations of certain 2-phenylvinylquinoline (2-styrylquinoline) and 2-furanylvinylquinoline derivatives

The present study describes the synthesis of 2-phenylvinylquinoline (styrylquinoline) and 2-furanylvinylquinoline derivatives and evaluation for their antiproliferative activities. (E)-2-Styrylquinolin-8-ol (14a) was inactive against a 3-cell line panel consisting of MCF-7 (Breast), NCI-H460 (Lung), and SF-268 (CNS). Replacement of the phenyl ring with 5-nitrofuran-2-yl group significantly enhanced antiproliferative activity in which (E)-2-(2-(5-nitrofuran-2-yl)vinyl)quinolin-8-ol (14i) and its 4-substituted derivatives 15–19 exhibited strong inhibitory effects against the growth of all three cancer cells. These compounds were further evaluated for their IC₅₀ against the growth of MCF-7, LNCaP, and PC3. Results indicated that a hydrogen bond donating oxime derivative 19a was more active than its hydrogen bond accepting methyloxime derivative 19b. For the inhibition of LNCaP, the potency decreased in an order 14i > 19a > 19b > 15 > 18 > 16. Compound 14i is the most active with an IC₅₀ value of 0.35 and 0.14 μM, respectively, against the growth of LNCaP and PC3 cancer cells. Therefore, compound 14i was evaluated by flow cytometric analysis for its effects on cell cycle distributions. Results indicated that 14i effectively induced cell cycle arrest at S phase for both cell lines, which consequently trigger late apoptosis for both LNCaP and PC3 cells.     

多功能的钙／钙调素依赖的蛋白激酶II介导的细胞信号传导

钙调素（Calmodulin,CaM）是一个特别的对钙敏感的蛋白,在钙信号传导通路中扮演重要角色钙／钙调素依赖性蛋白激酶（Calcium／calmodulin-dependent kinases（CaMKs））与荷尔蒙、神经迷质及其他信号引起的细胞反应相关、作为重要的第二信使,钙／钙调素依赖的蛋白激酶Ⅱ（CaM—KⅡ）是一类在细咆中无所不在的表达的蛋白激酶,能维持细胞内的钙浓度在很低的水平,再增加后续的特定的钙激动刺激。钙／钙调素依赖的簧白激酶Ⅱ独特的全酶结构和自我调节的性质使其对短暂的钙信号和胞内钙的变化能做出延长反应：本文从结构、合成、细胞分布、反应底物、生理功能等方面介绍了钙／钙调素依赖的蛋白激酶Ⅱ的激活对细胞信号传导的作用。     

Phenotype and functional evaluation of ex vivo generated antigen-specific immune effector cells with potential for therapeutic applications

Ex vivo activation and expansion of lymphocytes for adoptive cell therapy has demonstrated great success. To improve safety and therapeutic efficacy, increased antigen specificity and reduced non-specific response of the ex vivo generated immune cells are necessary. Here, using a complete protein-spanning pool of pentadecapeptides of the latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV), a weak viral antigen which is associated with EBV lymphoproliferative diseases, we investigated the phenotype and function of immune effector cells generated based on IFN-γ or CD137 activation marker selection and dendritic cell (DC) activation. These ex vivo prepared immune cells exhibited a donor- and antigen-dependent T cell response; the IFN-γ-selected immune cells displayed a donor-related CD4- or CD8-dominant T cell phenotype; however, the CD137-enriched cells showed an increased ratio of CD4 T cells. Importantly, the pentadecapeptide antigens accessed both class II and class I MHC antigen processing machineries and effectively activated EBV-specific CD4 and CD8 T cells. Phenotype and kinetic analyses revealed that the IFN-γ and the CD137 selections enriched more central memory T (Tcm) cells than did the DC-activation approach, and after expansion, the IFN-γ-selected effector cells showed the highest level of antigen-specificity and effector activities. While all three approaches generated immune cells with comparable antigen-specific activities, the IFN-γ selection followed by ex vivo expansion produced high quality and quantity of antigen-specific effector cells. Our studies presented the optimal approach for generating therapeutic immune cells with potential for emergency and routine clinical applications.